1. Field of the Invention
The present invention relates generally to vectors used in gene therapy. More specifically, the present invention relates to adeno-associated viruses and methods of producing said virus.
2. Description of the Related Art
Adeno-associated virus (AAV) is a single-stranded DNA virus of the family of Parvoviridae that has promising features as a vector for gene therapy. First, recombinant adeno-associated virus (rAAV) vectors can transduce terminally differentiated and non-dividing cells. Second, the lack of any apparent pathogenicity, low immunogenicity, relatively high stability of transgene expression, and the potential of targeted integration makes the rAAV superior over adenovirus and other viruses currently used in gene therapy trials. Last, a diversity of studies indicates that rAAV may provide a broad range of clinical applications to treat diseases including neurologic diseases, cancers and inherited monogenic defects, such as beta-thalassemia, sickle cell anemia, Fanconi anemia, chronic granulomatous disease, Gaucher disease, metachromatic leukodystrophy and cystic fibrosis, as well as acquired diseases, such as HIV infection and non-Hodgkin lymphoma.
The major limitation facing practical development of rAAV gene therapy is the difficulty in producing high titer rAAV. There are several factors which affect the ability to produce rAAV a t high titer, including transfection of the AAV genes (cap, rep and therapeutic genes) into host cells (4, 5), obtaining the optimal molar ratio of AAV DNA to helper adenovirus (6, 7, 8), and regulation between AAV rep and cap gene expression (9, 10, 11).
Recombinant adeno-associated virus (rAAV) is currently produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, which is required for rAAV replication and assembly, the virus is purified by CsCl gradients from total cell lygates (12, 13, 14). Because of the low efficiency of DNA transfection, it has previously been impossible to generate high titer rAAV (8, 15, 16, 17, 18, 19, 20).
As stated above, one of the limitations affecting the production of rAAV is determining the optimal molar ratio of the transfected AAV DNA and the Ad helper adenovirus. Pre-infection of host 293 cell with helper adenovirus and/or increasing the dose of helper adenovirus leads to a slight increase in the titer of rAAV (8). However, because adenovirus is cytolytic to 293 cells, it is not possible to achieve optimal production of rAAV before the cells are lysed by the adenovirus.
Regulation of the relative efficiency of the AAV rep and cap gene expression has been shown to significantly affect the production of rAAV. Overexpression of AAV rep78/68 proteins by substituting the p5 promoter with a strong heterologous promoter resulted in considerably lower yields of rAAV (6, 9, 10). In contrast, reduction of rep78/68 protein expression by using attenuated translation initiation codon ACG has resulted in much higher yields of rAAV (11). Replacement of the AAV p40 promoter with the strong cytomygalovirus early promoter can also lead to higher rAAV production (21).
A number of Ad genes, including the E1a, E1b, E2a, E4, and VAI RNA genes, are required for rAAV production (22, 23, 24, 25). The E1a gene product gerves as a transactivator, which leads to up-regulation of the transcriptional activity of numerous Ad genes, as well as the AAV rep and cap genes (22, 23). By interacting with E4, the E1b gene can facilitate the transport of viral mRNAs (25). The E4 gene, particularly open reading frame 6, is involved in facilitating AAV DNA replication. E2a and VAI RNA act to enhance the viral mRNA stability and the efficiency of translation, especially for AAV cap transcripts (25, 26). Two groups have demonstrated that the use of an adenoviral mini-plasmid containing only genes essential for rAAV provides sufficient helper function for growth of rAAV, resulting in relatively high titer rAAV (4, 6, 7). However, a large scale production of rAAV has not been successful because existing methods depend on low efficiency DNA transfection.
The Loxp sequence consists of two 13-bp inverted repeats separated by an 8-bp spacer (27). Cre recombinase can efficiently excise a sequence flanked by Loxp on both sides. Recently, the Loxp/Cre system has been successfully used to conditionally express a cytotoxic gene (26) and to control packaging of adenovirus (29, 30, 31). The conditional-packaging Ad helper virus grows as an E1 deleted adenovirus in 293 cells, but no virion is produced when this virus is grown in 293 cells that express Cre, since the Ad packaging sequence is flanked by Loxp and therefore, excised by the Cre protein. This virus still provides all functions for mini-AdAAV and mini-AdrAAV viral particle formation.
Thus, the prior art is deficient in efficient methods of high titer and large scale production of rAAV. The present invention fulfills this long-standing need and desire in the art.
The present invention discloses a method of producing large-scale rAAV stocks using co-infection of two recombinant adenoviruses. One encodes the GFP marker gene flanked by the terminal repeat ends of AAV, and the other encodes the AAV rep and cap genes. After heat inactivation of adenovirus, 1xc3x971010 efu/ml of rAAV-GFP were produced. To compare with conventional two plasmid co-transfection methods, 105 more rAAV can easily be produced with the novel method described herein. Two major improvements over previous methods are introduced in the present invention: (i) construction of recombinant AdAAV encoding rep and cap was done by DNA homologous recombination in bacterial hosts to avoid difficult generation of AdAAV in eukaryotic cells, and (ii) co-transfection of two recombinant adenoviruses instead of two plasmids plus adenovirus resulted in the possibility of large scale production of rAAV.
One object of the present invention is to provide a method of large-scale production resulting in high titers of rAAV using two recombinant adenoviruses instead of using an AAV plasmid to transmit the AAV genes into host cells.
In an embodiment of the present invention, there is provided a method of producing high titers of recombinant adeno-associated virus comprising a therapeutic gene (rAAV-Th), comprising the steps of: a) infecting cells with: (i) a recombinant helper adenovirus (AdAAV), wherein the AdAAV comprises an E1-deleted adenovirus genome, wherein the AdAAV comprises adeno-associated virus (AAV) rep and cap genes; and (ii) a recombinant adenovirus (AdrAVV-Th), wherein the recombinant adenovirus comprises a therapeutic gene (Th), wherein the Th is flanked by AAV ITR ends; b) purifying and titering viral particles from the cells, wherein the viral particles comprise recombinant AAV comprising the therapeutic gene (rAAV-Th), wherein high titers of the rAAV-Th are produced.
In another embodiment of the present invention, there is provided a method of producing high titers of recombinant adeno-associated virus comprising a therapeutic gene (rAAV-Th), comprising the steps of: a) infecting cells with: (i) a conditional-packageable adenoviral helper (hAd) vector, wherein the hAd vector comprises genes encoding adenoviral packaging functions, wherein the genes encoding the packaging functions are flanked by Loxp sequences; (ii) at least one recombinant adeno-associated virus vector, wherein the recombinant adeno-associated virus vector comprises adeno-associated virus (AAV) genes encoding rep and cap proteins, wherein the recombinant adeno-associated virus vector comprises a therapeutic gene (Th), wherein the therapeutic gene is flanked by adeno-associated virus (AAV) inverted terminal repeat (ITR) ends, and wherein the recombinant adeno-associated virus vector lacks genes encoding adenoviral packaging functions; whereby the infection produces adenoviral particles comprising a recombinant adeno-associated virus comprising the therapeutic gene (rAAV-Th); and b) purifying and titering the rAAV-Th, wherein high titers of the rAAV-Th are produced. When the conditional-packaging adenoviral helper vector comprises a gene encoding herpes simplex virus thymidine kinase, the above-described method may further comprise the step of: treating the infected cells with ganciclovir, whereby the treatment reduces contamination of the adenoviral particles by the conditional-packaging adenoviral helper vector.
In still yet another embodiment of the present invention, there is provided a recombinant adeno-associated virus (AdAAV), wherein the AdAAV comprises an E1-deleted adenovirus genome, wherein the AdAAV comprises adeno-associated virus (AAV) rep and cap genes. Furthermore, a mini-recombinant adeno-associated virus may be produced in which the adenovirus genome is deleted for all coding sequences other than those genes required for adeno-associated viral replication.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.